DESCRIPTION OF THE PRIOR ART
Hypusine, [(2S,9R)-2,11-diamino-9-hydroxy-7-azaundecanoic acid] or [N.sub..epsilon. -(4-amino-2-hydroxybutyl)lysine], an unusual naturally occurring amino acid, having the structure: ##STR2##
was first isolated from bovine brain extracts by Shiba et al. in 1971 [Biochim. Biophys. Acta., Vol. 244, pages 523-531 (1971)]. The molecule has two chiral centers, one at position 2 and one at position 9, each of which can be classified R or S by the Cahn-Ingold-Prelog method. The (2S,9R)-diastereomer (B), formed as a post-translational ##STR3##
modification of lysine, has been shown to occur on a precursor protein of the eukaryotic initiation factor "eIF-5A" (formerly called eIF-4D; the nomenclature for initiation factors having been revised) [Cooper et al., Proc. Natl. Acad. Sci. USA, Vol. 80, pages 1854-1857 (1983); and Safer, Eur. J. Biochem., Vol.186, pages 1-3 (1989)]. Deoxyhypusine, [2,11-diamino-7-azaundecanoic acid], has the following chemical structure: ##STR4##
The naturally occurring form has a chiral center at the 2-position in the (S) configuration.
The 17-kDa protein eIF-5A seems to be very highly conserved amongst many eukaryotic species including yeast and higher mammals, attesting to its importance from an evolutionary perspective. [Wohl, T., et. al., Mol. Gen. Genet., Vol. 241, 305-311 (1993); Magdolen, V. et al., Mol. Gen. Genet., Vol. 244, 646-652 (1994)]. In particular, the 12-amino acid region surrounding the hypusine residue, L-Ser-L-Thr-L-Ser-L-Lys-L-Thr-Gly-Hpu-L-His-Gly-L-His-L-Ala-L-Lys, SEQ ID NO:1 is extremely well-conserved across species. [Bartig, D. et al., Eur. J. Biochem., Vol. 204, 751-758 (1992)]. Hypusination of eIF--5A, or "maturation" of this protein, occurs as a post-translational event. [Park, M. H. et al., J. Biol. Chem., Vol. 257, 7217-7222 (1982)]. An aminobutyl group is first removed from spermidine and attached to Lys-50 of the human protein via deoxyhypusine synthase. [Park, M. H.; Wolff, E. C.; Abbruzzese, A.; Folk, J. E., Adv. Exp. Med. Biol. Vol. 250, 435-447 (1988); Wolff, E. C.; Park, M. H.; Folk, J. E. J. Biol. Chem., Vol. 265,4793-4799 (1990)]. Next, deoxyhypusine hydroxylase introduces the hydroxyl group at C-9 in the (R-) configuration [Park (1982)].
In the mid-1970's, eIF-5A was shown to stimulate ribosomal subunit joining and to enhance 80 S-bound Met-t-RNA reactivity with puromycin [Anderson et al., FEBS Lett., Vol. 76, pages 1-10 (1977); and Kemper et al., J. Biol. Chem., Vol. 251, pages 5551-5557 (1976)]. Later, in 1983, Cooper et al., supra, suggested that a hypusine-modified protein serves as an important initiation factor in all growing eukaryotic cells. In 1986, Park et al. [J. Biol. Chem., Vol. 261, pages 14515-14519 (1986)] isolated the eIF-5A protein from human red blood cells and elucidated the amino acid sequence surrounding the single hypusine residue, as Thr-Gly-Hpu-His-Gly-His-Ala-Lys. SEQ ID NO:6. In addition, because of the potential application to the control of HIV replication [Bevec et al., J. Proc. Natl. Acad. Sci. USA, Vol. 91, pages 10829-10833 (1994); and Ruhl et al. J. Cell Biol., Vol. 123, pages 1309-1320 (1994)], the synthesis of eIF-5A analogues is of great therapeutic significance.
The inhibitor of deoxyhypusine synthase, N.sup.1 -guanyl-1,7-diaminoheptane (CG.sub.7), inhibits the growth of CHO cells without affecting polyamine metabolism. [Park, M. H., et al., J. Biol. Chem., Vol. 269, 27827-27832 (1994)]. Site-directed mutagenesis experiments in which Lys-50 was replaced with arginine resulted in a nonfunctional protein in yeast cells; the arginine could not be modified to form hypusine [Schnier, J, et al., Mol. Cell. Biol., Vol. 11, 3105-3114 (1991)]. Furthermore, yeast cells that had the wild-type copy of the gene replaced with the mutant copy failed to grow. The precise role of the hypusine residue in eIF-5A activity remains elusive. While it is clear that the N-terminal methionine of the protein is replaced with an acetyl group, and that acetylation occurs at Lys-47, neither event seems critical to the protein's function [Klier, H. et al., FEBS Lett. Vol. 334, 360-364 (1993); Klier, H., et al., Biochemistry, Vol. 34, 14693-14702 (1995)].
Probably the most intriguing aspect regarding eIF-5A is its role in the replication of human immunodeficiency virus (HIV); eIF-5A is a transactivating factor during replication of HIV. [Ruhl, M., et al., J. Cell Biol., Vol.123, 1309-1320 (1993)]. The eIF-5A molecule binds to a complex formed between the Rev Response Element (RRE) in the Stem-Loop IIB of the viral mRNA and Rev, a viral protein that serves as a nuclear export signal. [Ruhl et al., supra]. Once eIF-5A binds to Rev-RRE, the now active eIF-5A-Rev-RRE complex is able to be exported from the nucleus; viral replication ensues. In experiments in which antisense nucleotides were used to prevent eIF-5A synthesis, viral replication was inhibited. [Gerace, L., Cell, Vol. 82, 341-344 (1995).] It has also been demonstrated in gel shift experiments that the hypusine- or deoxyhypusine-containing fragments were required for this binding of eIF-5A to Rev-RRE. In keeping with these observations, two issues become particularly intriguing. Bevec et al. have shown that Rev has domains which direct both nuclear import and nuclear export. Certain eIF-5A mutants, while capable of being transported into the nucleus and binding to Rev-RRE, actually prevent nuclear export and, thus, viral replication. [Bevec, D., et al., Science, Vol. 271,1858-1860 (1996); Junker, U.; et al., Hum. Gene Ther., Vol. 7, 1861-1869 (1996)].
The observations that eIF-5A is required for both mitotic events and HIV viral replication and that immature eIF-5A must be deoxyhypusinated or hypusinated for activity render inhibition of eIF-5A deoxyhypusination or hypusination an interesting target in therapeutic strategies for anticancer and antiviral drug development. Another potential antiviral strategy involves identifying the basic platform within eIF-5A responsible for nuclear import that will permit Rev-RRE binding but not nuclear export of viral message.
In order to study the above-described biochemical events and to develop therapeutic strategies for anticancer and antiviral drug development, there is a need for synthetic methodology for accessing model peptides containing hypusine and deoxyhypusine. A reagent and method for producing peptides incorporating hypusine are disclosed in a co-pending application, U.S. Ser. No. 09/136,270, entitled "Hypusine Reagent for Peptide Synthesis," filed on Aug. 19, 1998, as a continuation-in-part application of U.S. Ser. No. 08/962,300, filed Oct. 31, 1997, which disclosure is incorporated herein by reference. Novel peptides incorporating hypusine produced via the method of the present invention are disclosed in a co-pending application, U.S. Ser. No. 09/136,472,entitled "Hypusine Peptides," filed on Aug. 19, 1998, as a continuation-in-part application of U.S. Ser. No. 08/975,656, filed Nov. 21, 1997, which disclosure is incorporated herein by reference. The hypusine reagent and peptides are also disclosed in Bergeron, R. J. et al., J. Org. Chem., Vol. 62, 3285-3290 (1997), which disclosure is incorporated herein by reference. It is an object of the present invention to provide novel peptides incorporating deoxyhypusine and a reagent and method for their synthesis.